Methionine in proteins defends against oxidative stress

Abstract

A variety of reactive oxygen species react readily with methionine residues in proteins to form methionine sulfoxide, thus scavenging the reactive species. Most cells contain methionine sulfoxide reductases, which catalyze a thioredoxin-dependent reduction of methionine sulfoxide back to methionine. Thus, methionine residues may act as catalytic antioxidants, protecting both the protein where they are located and other macromolecules. To test this hypothesis directly, we replaced 40% of the methionine residues in Escherichia coli with norleucine, the carbon-containing analog, in which the sulfur of methionine is substituted by a methylene group (-CH2-). The intracellular free methionine and S-adenosylmethionine pools were not altered, nor was the specific activity of the key enzyme, glutamine synthetase. When unstressed, both control and norleucine-substituted cells survived equally well at stationary phase for at least 32 h. However, oxidative stress was more damaging to the norleucine-substituted cells. They died more rapidly than control cells when challenged by hypochlorite, hydrogen peroxide, or ionizing radiation. One of the most abundant proteins in the cell, elongation factor Tu, was found to be more oxidatively modified in norleucine-substituted cells, consistent with loss of the antioxidant defense provided by methionine residues. The results of these studies support the hypothesis that methionine in protein acts as an endogenous antioxidant in cells.

Figure 1.

Scheme of the antioxidant defense system created by cyclic oxidation and reduction of Met residues. Reduced forms of the proteins carry the subscript “red” and oxidized forms carry “ox.” Reading from left to right, ROS is intercepted by a Met residue, which is oxidized to MetO. MetO is reduced back to Met by msr, with the formation of a disulfide bond. The oxidized msr is reduced by thioredoxin, which now carries the disulfide bond. It is reduced by thioredoxin reductase, which, in mammals, contains a selenocysteine residue that is oxidized, forming a selenocysteine-cysteine bond. This disulfide analog is then reduced by NADPH. The net result is that ROS is reduced at the expense of NADPH.

Figure 2.

Growth in Nle-containing medium reduces protein Met content without affecting the free Met pool. A) Met and Nle content of cells. B) Free Met content.

Figure 3.

Growth in Nle-containing medium does not affect S-adenosylmethionine pool size nor the activity of glutamine synthetase. A) S-adenosylmethionine content. B) Glutamine synthetase activity.

Figure 4.

Nle-containing cells are more susceptible to killing by oxidative stresses. A) Viability after 1 h exposure to hypocholorite. B, C) Viability after exposure to 5 mM hydrogen peroxide. D) Viability after 10–40 Gy irradiation exposure at 37°C. E) Viability after 100–1200 Gy irradiation exposure at 0°C.

Figure 5.

Protein carbonylation. A) Western blot. Lanes 1–2: oxidized and nonoxidized glutamine synthetase standards. Lanes 3–10: whole-cell SDS extracts. Cells were sampled after 1 h at 37°C in the absence or presence of 5 μM (control cells) or 4 μM (Nle cells) hypochlorite, 5 mM hydrogen peroxide, or 10 Gy irradiation. Lanes 11–12: Duplicates of lanes 3 and 7 but without 2,4-dintriophenylhydrazine exposure as a control for specificity of carbonyl labeling. B) Quantitation of the results. Bars show average and sd of 2 independent experiments.

Figure 6.

MetO content, shown as the percentage of the sum of Met + MetO. A) MetO content in hypochlorite-exposed cells. B) Cell viability as a function of MetO content in hypochlorite-exposed cells (from A and Fig. 4A). C, D) MetO content in control cells (C) and cells exposed to hydrogen peroxide (D). E) MetO content in irradiated cells.

Figure 7.

Topography of Met in elongation factor Tu. A) Schematic diagram of EF-Tu. Stick model shows the 10 Met residues in red with their sequence numbers. Six (112, 139, 151, 260, 349, and 351) are surface exposed. Domain 1 is light blue, domain 2 dark blue, and domain 3 green. Nucleotide GDP is rendered as a ball-and-stick model; Mg²⁺ is shown as a purple sphere. B) CPK diagram of EF-Tu. For clarity, only domains 1 and 3 are shown; colors as in A. Within Met residues, sulfur atoms are yellow, carbon atoms are gray, and oxygen atoms are red. An additional 2 Met residues (91 and 98) are solvent accessible. Figures were generated with DSViewerPro (Accelrys, San Diego, CA, USA) from the coordinates of Song et al. , taken from the Protein Data Bank entry 1EFC.